Correlation between IL1β expression level and morphological parameters proves the usefulness of morphology measures to predict the degree of activation of microglial cells

It is well known that microglial cells undergo an important change in morphology upon activation, so that form and function are intimately related. Upon activation, microglia cell body enlarges, its ramifications shortens and become thicker. In parallel, a variety of cytokines and inflammatory mediators such as IL1β are released. However the activation process is not all-or-nothing. Rather, cells in subtle activation states or in a deactivation process can occur, so intermediate not obvious phenotypes may appear. Thus, we aimed to correlate the expression level of a well-defined marker of activation, IL1β, with different morphological parameters. To do so, we used an intracerebroventricular injection of neuraminidase to produce an acute inflammation in rats. Brain sections were double-stained with IBA1 to have an image of the whole cell and its ramifications, and with IL1β to assess the level of activation. Images were captured from septofimbria (close to the injection site) and from the hypothalamus. A ratio of IL1β-positive pixels to IBA1-positive pixels was used to estimate the level of IL1β expression for each cell. Single microglial cell images were processed with ImageJ software to obtain outlined and filled shapes, which were used to obtain (by means of FracLac plug in) the following morphological parameters: fractal dimension, lacunarity, area, density and perimeter. All parameters showed a significant correlation with the level of expression of IL1β. This occurred in cells sampled from the two brain areas studied. Density, lacunarity and perimeter resulted as the best predictor parameters of activation, that is, those with a better correlation with the level of expression of IL1β. Area, an extensively used parameter to assess microglial activation, presented the least significant correlation. Thus, objectively measured morphological parameters correlate with the level of expression of IL1β, and could therefore be used as predictors of the activation level of microglial cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Morphological traits of surveillant/activated microglia during an experimentally induced acute neuroinflammatory process

Poster en CongresoSeveral studies show that morphological changes of microglia over the course of inflammation are tightly coupled to function. However the progressive transformation into activated microglia is poorly characterized. AIMS: This study aimed to establish a spatiotemporal correlation between quantifiable morphological parameters of microglia and the spread of an acute ventricular inflammatory process. METHODS: Inflammation was induced by a single injection of the enzyme neuraminidase within the lateral ventricle of rats. Animals were sacrificed 2, 4 and 12 hours after injection. Coronal slices were immunostained with Iba1 to label microglia and with IL1β to delimit the spread of inflammation. Digital images were obtained by scanning the labelled sections. Single microglia images were randomly selected from periventricular areas of caudate putamen, hippocampus and hypothalamus. FracLac for ImageJ software was used to measure the following morphological parameters: fractal dimension, lacunarity, area, perimeter and density. RESULTS: Significant differences were found in fractal dimension, lacunarity, perimeter and density of microglia cells of neuraminidase injected rats compared to sham animals. However no differences were found in the parameter “area”. In hipoccampus there was a delay in the significant change of the measured parameters. These morphological changes correlated with IL1β-expression in the same areas. CONCLUSIONS: Ventricular inflammation induced by neuraminidase provokes quantifiable morphological changes in microglia restricted to areas labelled with IL1β. Morphological parameters of microglia such as fractal dimension, lacunarity, perimeter and density are sensitive and valuable tools to quantify activation. However, the extensively used parameter “area” did not change upon microglia activation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Grant: Junta de Andalucía (Spain) P11-CVI-07637 Ibima, Andalucía Tech, UM

Neuraminidase-induced neuroinflammation causes anxiety and microgliosis in the amygdala

An intracerebroventricular (ICV) injection of neuraminidase (NA) within the lateral ventricles originates an acute event of neuroinflammation, which is solved to a great extent after two weeks. Recently, neurological problems or behavioral alterations have been associated with neuroinflammation. Although the majority of them fade along with inflammation resolution, the possibility of long-term sequelae should be taken into consideration. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Initially, rats were ICV injected with NA or saline. Two or 10 weeks later they were made to perform a series of neurological tests and behavioral evaluations (open field test). The neuroinflammation status of the brain was studied by immunohistochemistry and qPCR. While no neurological alterations were found, the open field test revealed an increased anxiety state 2 weeks after NA administration, which was not observed after 10 weeks. In accordance with this behavioral findings, an overexpression of the molecular pattern receptor TLR4 was revealed by qPCR in hypothalamic tissue in NA treated animals after 2 weeks of ICV, but not after 10 weeks. Moreover, histological studies showed a microgliosis in the amygdala of NA injected rats 2 weeks post-ICV, as well as a slightly activated state evidenced by morphometric parameters of these cells. These histological findings were not present 10 weeks after the ICV injection. These results suggest that NA-induced neuroinflammation might cause anxiety, with no neurological manifestations, in the medium term, along with a mild microglial activation in amygdala. Such symptoms seem to revert, as they were not detected 10 weeks after NA administration.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Enhanced stress response in rats that suffered acute neuroinflammation induced by neuraminidase three months before

Microglial cells are protagonists in neuroinflammatory processes and their activation is a notorious feature of such events. In acute inflammation, microglial cells return to their basal surveillant state in few days. However, sometimes they evolve towards a primed state, characterized by hypersensitivity to new stimuli and an exacerbated response which may jeopardize brain functions. Because the hypothalamus is a pivotal hub for neuroendocrine and autonomic functions, we have been exploring evidences of microglial priming in this region and its consequences. We used a model of acute ventricular neuroinflammation consisting in the intracerebroventricular (ICV) injection of neuraminidase (NA). This enzyme is found in the cover of neurotropic bacteria and viruses, e.g. influenza, mumps or measles viruses, thus mimicking a brain infection. Three months after inducing neuroinflammation with NA to rats, an acute stressor was applied to investigate the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the stress response elicited, as well as the inflammatory activation of hypothalamic microglial cells. The acute stressor was forced swimming for 6 minutes. Afterwards, blood samples were retrieved to determine corticosterone levels by ELISA, and the brains extracted to analyze microglial cells in histological sections by immunohistochemistry with IBA1 and inflammatory markers by qPCR. Stressed rats previously injected with NA had increased levels of corticosterone compared with control rats that were equally stressed but had been ICV injected with saline. Also, qPCR studies in hypothalamic tissue revealed that NA treated rats presented an increased expression of the genes for the inflammasome protein NLR family pyring domain containing 3 (NLRP3) and the microglial marker IBA1. Concomitantly, the morphological analysis of microglial cells located in the paraventricular nucleus (PVN) showed a morphological bias towards a slightly activated state in microglia...Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech